Cryopreservation of mammalian reproductive cells is an essential technique in IVF (in vitro fertilization) clinics. Oocytes and embryos are routinely frozen and preserved for use at a later time. Patients who undergo therapeutic procedures that can place their fertility at risk, such as chemotherapy, have the option of preserving their oocytes for future use through IVF techniques. Additionally, fertilized embryos are often more needed for one cycle of IVF treatment. The rest of fertilized embryos are usually cryopreserved for future use.
Techniques of oocyte/embryo cryopreservation are classified into two categories, slow freezing and fast freezing (i.e., vitrification). Slow freezing is a well-established technique developed during the early 1970s, which makes use of programmable sequences, or controlled cooling rates. Vitrification or fast freezing is a more effective cryopreservation method, first reported in 1985 (see W. F. Rall and G. M. Fahy, “Ice-free cryopreservation of mouse embryos at −196 degrees C. by vitrification,” Nature, vol. 313, no. 6003, pp. 573-5, 1985). Vitrification is considered superior to slow freezing, because it vitrifies the oocyte/embryo with no crystal formation during freezing. The addition of cryoprotectants in vitrification increases the embryo viscosity, and makes the vitrified embryos syrupy. When directly freezing oocytes/embryos in liquid nitrogen, the syrupy content inside the cell forms amorphous ice instead of ice crystals, which minimize the vital damage to the cell during freezing.
Oocyte/embryo vitrification is done manually in IVF clinics. An operator looks though the microscope eyepieces and manipulates oocytes/embryos using a micropipette. An oocyte/embryo is first taken out from the culture dish and washed with the equilibrium solution (ES) and a series of vitrification solution (VS). Within each step, the control of timing has been proven critical. After the washing steps, the processed oocyte/embryo is placed onto a vitrification straw or into a cryo-pipette. The volume of solution remained around the oocyte/embryo on the straw must be minimal in order to ensure a high cooling rate. The vitrification straw is then plunged into liquid nitrogen for freezing and long-term cryopreservation. A number of different commercial vitrification solutions and protocols exist; however, the core steps are largely the same. All the protocols involve multiple washing steps with ES and VS, placing the vitrified oocytes/embryos on vitrification straws, and freezing the vitrification straws in liquid nitrogen.
Manual operation for oocyte/embryo vitrification is a demanding and tedious task, for the following reasons: (1) washing oocytes/embryos with the highly viscous VS causes osmotic shock to the cells, and osmotic shock can be a major cause for cell damage; (2) most antifreezing solutes (e.g., DMSO) are toxic to ooctytes/embryos. Therefore, the washing time in toxic VS is critical but can be difficult to strictly control; (3) because of their small size (about 150 μm), oocytes/embryos can be difficult to detect and manipulate, especially when the medium surrounding the cells is dynamically changing during micropipette aspiration and dispensing; (4) the manual process has stringent skill requirements, and success rate and cell survival rate vary across different operators.
Heo et al. developed a microfluidic platform to control the loading of cryoprotectants for oocyte cryopreservation (see Y. Heo, H. Lee, B. Hassell, et al., “Controlled loading of cryoprotectants (CPAs) to oocyte with linear and complex CPA profiles on a microfluidic platform,” Lab on a Chip, vol. 11, no. 20, pp. 3530-7, 2011). When an oocyte/embryo is ‘parked’ at a position of the microfluidic platform, one side of the oocyte/embryo directly faces the vitrification solutions while the other side does not. Accordingly, the oocyte/embryo is not exposed to VS as uniformly as in the standard manual protocols. In practice, parking embryos in a container or preset location can bring about significant difficulties for retrieving embryos after washing in VS. The timing of imposing oocytes/embryos to VS is critical because the cryoprotectants in VS can impose toxic effects on embryos if they cannot be retrieved in time.
Genea Biomedx Inc. developed an automated instrument for oocyte/embryo vitrification (U.S. Pat. Appl. Publ. No. 2013/0137080). The Genea system requires a user to manually transfer oocytes/embryos into an array of wells termed ‘pods’ with one oocyte/embryo in each well. In present vitrification protocols used in IVF clinics, an oocyte/embryo is moved in and out of different vitrification solutions. Differently, oocytes/embryos in the Genea system stay in the wells while vitrification solutions are dispensed into and aspirated out of the wells. Since the oocyte/embryo always sits on the bottom of the well, the cell surface in contact with the well bottom cannot be exposed to vitrification solutions as uniformly as the rest of the cell surface. Furthermore, the Genea system dispenses and aspirates fluids based on volume control without monitoring cell position; therefore, the technology cannot meet the minimal volume requirement to achieve a high cooling rate.
Similar to Genea Biomedx's automated vitrification system, patent application publication numbers WO2013020032 by Samuel S. Kim et al, US2011/0207112 by Fred Burbank et al, and WO2013098825 by Amir Arav also disclosed methods of parking oocytes/embryos in preset locations and changing VS. WO2013020032 by Samuel S. Kim et al. describes an automated device comprising a cryoprotectant holder, a cryoprotectant dispenser, and a sample holder oriented to allow a sample to be in contact with cryoprotectant from said cryoprotectant dispenser. In this device, oocytes or embryos are kept in a sample holder (e.g., electron microscopy grid) throughout the entire procedure, while the VS are dispensed in and drained out for washing the sample.
In U.S. Pat. Appl. Publ. No. 2011/0207112 by Fred Burbank et al., one or more oocytes or embryos are positioned in a processing container, the processing container being configured to allow fluid to flow into and out of the processing container, where two or more fluids flow into and out of the container.
WO20130988205 by Amir Arav disclosed a device comprising a draining zone and a capillary draining element. The draining zone is configured to hold a reproductive biological sample. The capillary draining element, whose opening is within the draining zone, is configured to drain liquid away from the draining zone while a reproductive cellular constituent of the reproductive cellular portion remains within the draining zone.
Different from the presently used vitrification protocols by placing vitrified oocyte/embryos to a straw-like carrier, US Pat. Publ. Nos. 2013/0157362 by Fuliang Du et al., 2002/0009704 by Xiangzhong Yang et al., and 2012/0251999 by Utkan Demirci et al. disclosed methods for generating micro/nano droplets of VSs comprising the biological sample, and freezing the droplets directly in liquid nitrogen.
Milton Chin disclosed in US Pat. Appl. Publ. Nos. 2009/0186405 and 2009/0123996 device designs for improving the chilling rate in liquid nitrogen and achieving the self-sealing function for storage of vitrified specimens in liquid nitrogen.
Ru et al. in Chinese Pat. Appl. Publ. No. 202918907U disclosed a semi-automated system for embryo vitrification. The system requires several key steps of manual input to conduct embryo pick-and-place. In paragraph 0056, the system requires the operator to obtain the micropipette tip position by observing from the microscope and input the position information into computer. Similar human inputs are required (see paragraphs 0062, 0070, and in 0072). The automation part is only about using robotic manipulator to pick up an embryo, move to a preset location, and place the embryo into vitrification solutions. There is no computer vision algorithm to automatically detect embryos and micropipette tip. Moreover, the technique of single cell pick-and-place with a robotic system is open knowledge, such as disclosed by Z. Lu, C. Moraes, G. Ye, C. A. Simmons, and Y. Sun in “Single cell deposition and patterning with a robotic system,” PLoS ONE, Vol., 5, e13542, 2010. Additionally, the system disclosed in CN202918907U can only work with a specific Cryotip® method (see paragraph 0086), which is limited for general applications. Without washing embryos in vitrification solutions, the system cannot be used for other protocols (e.g., Kitazato's Cryotop method, and Irvine vitrification protocol) that require embryos to be washed for at least one time.
What is needed is an automated vitrification system designed to automatically process oocytes/embryos and other cell types with vitrification processing solutions, automatically place vitrified oocytes/embryos to vitrification devices (e.g., Cryotop, Cryotip®, Cryoloop, etc.), remove excessive medium from the vitrified oocytes/embryos on the devices, automatically seal the vitrification devices with caps on a sealing machine, and freeze in an automated liquid nitrogen storage tank/system.